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- PDB-9f26: Crystal structure of the PriS_PriL-Rpa2WH ternary complex from P.... -

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Basic information

Entry
Database: PDB / ID: 9f26
TitleCrystal structure of the PriS_PriL-Rpa2WH ternary complex from P. abyssi
Components
  • DNA primase large subunit PriL
  • DNA primase small subunit PriS
  • RPA32 subunit of the hetero-oligomeric complex involved in homologous recombination
KeywordsDNA BINDING PROTEIN / Replication protein A / ssDNA-Binding protein
Function / homology
Function and homology information


primosome complex / : / DNA-directed RNA polymerase complex / Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases / 4 iron, 4 sulfur cluster binding / nucleic acid binding / chromosome, telomeric region / metal ion binding
Similarity search - Function
DNA primase large subunit PriL / DNA primase small subunit PriS / Replication factor A protein-like / DNA primase, small subunit, eukaryotic/archaeal / DNA primase, small subunit / DNA primase small subunit / DNA primase large subunit, eukaryotic/archaeal / Eukaryotic and archaeal DNA primase, large subunit / OB-fold nucleic acid binding domain, AA-tRNA synthetase-type / OB-fold nucleic acid binding domain ...DNA primase large subunit PriL / DNA primase small subunit PriS / Replication factor A protein-like / DNA primase, small subunit, eukaryotic/archaeal / DNA primase, small subunit / DNA primase small subunit / DNA primase large subunit, eukaryotic/archaeal / Eukaryotic and archaeal DNA primase, large subunit / OB-fold nucleic acid binding domain, AA-tRNA synthetase-type / OB-fold nucleic acid binding domain / Winged helix-like DNA-binding domain superfamily / Nucleic acid-binding, OB-fold
Similarity search - Domain/homology
RPA32 subunit of the hetero-oligomeric complex involved in homologous recombination / DNA primase large subunit PriL / DNA primase small subunit PriS
Similarity search - Component
Biological speciesPyrococcus abyssi (archaea)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 3.501 Å
AuthorsMadru, C. / Legrand, P. / Haouz, A. / Sauguet, L.
Funding support France, 2items
OrganizationGrant numberCountry
Agence Nationale de la Recherche (ANR)ARCHAPRIM France
Fondation pour la Recherche Medicale (FRM) France
CitationJournal: Nat Commun / Year: 2024
Title: Communication between DNA polymerases and Replication Protein A within the archaeal replisome.
Authors: Markel Martínez-Carranza / Léa Vialle / Clément Madru / Florence Cordier / Ayten Dizkirici Tekpinar / Ahmed Haouz / Pierre Legrand / Rémy A Le Meur / Patrick England / Rémi Dulermo / J ...Authors: Markel Martínez-Carranza / Léa Vialle / Clément Madru / Florence Cordier / Ayten Dizkirici Tekpinar / Ahmed Haouz / Pierre Legrand / Rémy A Le Meur / Patrick England / Rémi Dulermo / J Iñaki Guijarro / Ghislaine Henneke / Ludovic Sauguet /
Abstract: Replication Protein A (RPA) plays a pivotal role in DNA replication by coating and protecting exposed single-stranded DNA, and acting as a molecular hub that recruits additional replication factors. ...Replication Protein A (RPA) plays a pivotal role in DNA replication by coating and protecting exposed single-stranded DNA, and acting as a molecular hub that recruits additional replication factors. We demonstrate that archaeal RPA hosts a winged-helix domain (WH) that interacts with two key actors of the replisome: the DNA primase (PriSL) and the replicative DNA polymerase (PolD). Using an integrative structural biology approach, combining nuclear magnetic resonance, X-ray crystallography and cryo-electron microscopy, we unveil how RPA interacts with PriSL and PolD through two distinct surfaces of the WH domain: an evolutionarily conserved interface and a novel binding site. Finally, RPA is shown to stimulate the activity of PriSL in a WH-dependent manner. This study provides a molecular understanding of the WH-mediated regulatory activity in central replication factors such as RPA, which regulate genome maintenance in Archaea and Eukaryotes.
History
DepositionApr 22, 2024Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 4, 2024Provider: repository / Type: Initial release
Revision 1.1Jan 22, 2025Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: DNA primase small subunit PriS
B: DNA primase large subunit PriL
C: RPA32 subunit of the hetero-oligomeric complex involved in homologous recombination
hetero molecules


Theoretical massNumber of molelcules
Total (without water)118,1514
Polymers118,0863
Non-polymers651
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area3750 Å2
ΔGint-15 kcal/mol
Surface area29420 Å2
MethodPISA
Unit cell
Length a, b, c (Å)63.815, 101.505, 177.805
Angle α, β, γ (deg.)90, 90, 90
Int Tables number19
Space group name H-MP212121

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Components

#1: Protein DNA primase small subunit PriS / DNA primase 41 kDa subunit / Pabp41 / p41


Mass: 40662.637 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: DNA primase small subunit PriS / Source: (gene. exp.) Pyrococcus abyssi (archaea) / Gene: priS, priA, PYRAB01820, PAB2236 / Production host: Escherichia coli BL21(DE3) (bacteria)
References: UniProt: Q9V292, Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases
#2: Protein DNA primase large subunit PriL / DNA primase 46 kDa subunit / Pabp46 / p46


Mass: 45548.426 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pyrococcus abyssi (archaea) / Gene: priL, priB, PYRAB01830, PAB2235 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q9V291
#3: Protein RPA32 subunit of the hetero-oligomeric complex involved in homologous recombination


Mass: 31874.826 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: RPA2 WH domain / Source: (gene. exp.) Pyrococcus abyssi (archaea) / Gene: PAB2165 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q9V1Z1
#4: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY
Has protein modificationN

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 4.02 Å3/Da / Density % sol: 69 %
Crystal growTemperature: 291 K / Method: vapor diffusion, hanging drop / pH: 7.1 / Details: 60% v/vTacsimate 0.1M Bis-Tris prop 7

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: SOLEIL / Beamline: PROXIMA 1 / Wavelength: 0.978565 Å
DetectorType: DECTRIS EIGER X 16M / Detector: PIXEL / Date: Jul 23, 2021 / Details: KB Mirrors
RadiationMonochromator: Si111 / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.978565 Å / Relative weight: 1
ReflectionResolution: 3.5→48.8 Å / Num. obs: 201145 / % possible obs: 99.9 % / Redundancy: 13.2 % / Biso Wilson estimate: 123.9 Å2 / CC1/2: 1 / Rpim(I) all: 0.036 / Rrim(I) all: 0.131 / Rsym value: 0.126 / Net I/σ(I): 9.5
Reflection shellResolution: 3.5→3.59 Å / Redundancy: 13.4 % / Mean I/σ(I) obs: 0.3 / Num. unique obs: 14465 / CC1/2: 0.2 / Rpim(I) all: 4.24 / Rrim(I) all: 15.624 / Rsym value: 15.026 / % possible all: 99.5

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Processing

Software
NameVersionClassification
BUSTER2.10.4refinement
XDSVERSION Feb 5, 2021 BUILT=20210323data reduction
XDSVERSION Feb 5, 2021 BUILT=20210323data scaling
STARANISO2.3.77data scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 3.501→48.8 Å / Cor.coef. Fo:Fc: 0.952 / Cor.coef. Fo:Fc free: 0.938 / Cross valid method: THROUGHOUT / SU Rfree Blow DPI: 0.81
RfactorNum. reflection% reflectionSelection details
Rfree0.2555 473 -RANDOM
Rwork0.2345 ---
obs0.2356 8875 58.5 %-
Displacement parametersBiso mean: 184.46 Å2
Baniso -1Baniso -2Baniso -3
1-16.4151 Å20 Å20 Å2
2--9.6578 Å20 Å2
3----26.0729 Å2
Refine analyzeLuzzati coordinate error obs: 0.62 Å
Refinement stepCycle: LAST / Resolution: 3.501→48.8 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms5289 0 1 0 5290
Refine LS restraints
Refine-IDTypeDev idealNumberRestraint functionWeight
X-RAY DIFFRACTIONt_bond_d0.0055394HARMONIC2
X-RAY DIFFRACTIONt_angle_deg0.767250HARMONIC2
X-RAY DIFFRACTIONt_dihedral_angle_d1999SINUSOIDAL2
X-RAY DIFFRACTIONt_gen_planes913HARMONIC5
X-RAY DIFFRACTIONt_it5394HARMONIC10
X-RAY DIFFRACTIONt_chiral_improper_torsion670SEMIHARMONIC5
X-RAY DIFFRACTIONt_utility_distance4HARMONIC1
X-RAY DIFFRACTIONt_ideal_dist_contact3758SEMIHARMONIC4
X-RAY DIFFRACTIONt_omega_torsion2.32
X-RAY DIFFRACTIONt_other_torsion17.76
LS refinement shellResolution: 3.501→3.67 Å
RfactorNum. reflection% reflection
Rfree0.4623 17 -
Rwork0.3307 --
obs--19.91 %
Refinement TLS params.

Refine-ID: X-RAY DIFFRACTION

IDL11 (°2)L12 (°2)L13 (°2)L22 (°2)L23 (°2)L33 (°2)S11 (Å °)S12 (Å °)S13 (Å °)S21 (Å °)S22 (Å °)S23 (Å °)S31 (Å °)S32 (Å °)S33 (Å °)T11 (Å2)T12 (Å2)T13 (Å2)T22 (Å2)T23 (Å2)T33 (Å2)Origin x (Å)Origin y (Å)Origin z (Å)
17.0777-0.1141-0.72346.6677-1.51592.04260.298-0.54420.0769-0.5442-0.0850.30660.07690.3066-0.2129-0.304-0.0462-0.1302-0.304-0.0908-0.30419.63532.6294-43.0832
28.3154-0.91282.06822.0976-2.64338.31540.19250.38980.54420.38980.1013-0.48930.5442-0.4893-0.29380.268-0.152-0.1520.3040.152-0.30439.8896-18.9752-12.5025
38.30262.9104-2.09677.8823-2.91045.9229-0.12540.5442-0.54420.54420.02070.4165-0.54420.41650.1047-0.24850.1411-0.0522-0.2659-0.05130.30351.998620.6677-39.1252
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection detailsAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1{ A|* }A0 - 345
2X-RAY DIFFRACTION1{ A|* }A901
3X-RAY DIFFRACTION2{ B|* }B1 - 211
4X-RAY DIFFRACTION3{ C|* }C189 - 268

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